Klebsiella pneumoniae carbapenemase variant 44 acquires ceftazidime-avibactam resistance by altering the conformation of active-site loops.

Klebsiella pneumoniae carbapenemase 2 (KPC-2) is an important source of drug resistance as it can hydrolyze and inactivate virtually all ß-lactam antibiotics. KPC-2 is potently inhibited by avibactam via formation of a reversible carbamyl linkage of the inhibitor with the catalytic serine of the enzyme. However, the use of avibactam in combination with ceftazidime (CAZ-AVI) has led to the emergence of CAZ-AVI-resistant variants of KPC-2 in clinical settings. One such variant, KPC-44, bears a 15 amino acid duplication in one of the active-site loops (270-loop). Here, we show that the KPC-44 variant exhibits higher catalytic efficiency in hydrolyzing ceftazidime, lower efficiency toward imipenem and meropenem, and a similar efficiency in hydrolyzing ampicillin, than the WT KPC-2 enzyme. In addition, the KPC-44 variant enzyme exhibits 12-fold lower AVI carbamylation efficiency than the KPC-2 enzyme. An X-ray crystal structure of KPC-44 showed that the 15 amino acid duplication results in an extended and partially disordered 270-loop and also changes the conformation of the adjacent 240-loop, which in turn has altered interactions with the active-site omega loop. Furthermore, a structure of KPC-44 with avibactam revealed that formation of the covalent complex results in further disorder in the 270-loop, suggesting that rearrangement of the 270-loop of KPC-44 facilitates AVI carbamylation. These results suggest that the duplication of 15 amino acids in the KPC-44 enzyme leads to resistance to CAZ-AVI by modulating the stability and conformation of the 270-, 240-, and omega-loops.

Evaluation of Tebipenem Hydrolysis by ß-Lactamases Prevalent in Complicated Urinary Tract Infections.

Tebipenem pivoxil is the first orally available carbapenem antibiotic and has been approved in Japan for treating ear, nose, and throat and respiratory infections in pediatric patients. Its active moiety, tebipenem, has shown potent antimicrobial activity in vitro against clinical isolates of Enterobacterales species from patients with urinary tract infections (UTIs), including those producing extended-spectrum ß-lactamases (ESBLs) and/or AmpC ß-lactamase. In the present study, tebipenem was tested for stability to hydrolysis by a set of clinically relevant ß-lactamases, including TEM-1, AmpC, CTX-M, OXA-48, KPC, and NDM-1 enzymes. In addition, hydrolysis rates of other carbapenems, including imipenem, meropenem, and ertapenem, were determined for comparison. It was found that, similar to other carbapenems, tebipenem was resistant to hydrolysis by TEM-1, CTX-M, and AmpC ß-lactamases but was susceptible to hydrolysis by KPC, OXA-48, and NDM-1 enzymes with catalytic efficiency values (kcat/Km) ranging from 0.1 to 2 × 106 M-1s-1. This supports the reported results of antimicrobial activity of tebipenem against ESBL- and AmpC-producing but not carbapenemase-producing Enterobacterales isolates. Considering that CTX-M and AmpC ß-lactamases represent the primary determinants of multidrug-resistant complicated UTIs (cUTIs), the stability of tebipenem to hydrolysis by these enzymes supports the utility of its prodrug tebipenem, tebipenem pivoxil hydrobromide (TBP-PI-HBr), as an oral therapy for adult cUTIs.

Antagonism between substitutions in ß-lactamase explains a path not taken in the evolution of bacterial drug resistance.

CTX-M ß-lactamases are widespread in Gram-negative bacterial pathogens and provide resistance to the cephalosporin cefotaxime but not to the related antibiotic ceftazidime. Nevertheless, variants have emerged that confer resistance to ceftazidime. Two natural mutations, causing P167S and D240G substitutions in the CTX-M enzyme, result in 10-fold increased hydrolysis of ceftazidime. Although the combination of these mutations would be predicted to increase ceftazidime hydrolysis further, the P167S/D240G combination has not been observed in a naturally occurring CTX-M variant. Here, using recombinantly expressed enzymes, minimum inhibitory concentration measurements, steady-state enzyme kinetics, and X-ray crystallography, we show that the P167S/D240G double mutant enzyme exhibits decreased ceftazidime hydrolysis, lower thermostability, and decreased protein expression levels compared with each of the single mutants, indicating negative epistasis. X-ray structures of mutant enzymes with covalently trapped ceftazidime suggested that a change of an active-site Ω-loop to an open conformation accommodates ceftazidime leading to enhanced catalysis. 10-µs molecular dynamics simulations further correlated Ω-loop opening with catalytic activity. We observed that the WT and P167S/D240G variant with acylated ceftazidime both favor a closed conformation not conducive for catalysis. In contrast, the single substitutions dramatically increased the probability of open conformations. We conclude that the antagonism is due to restricting the conformation of the Ω-loop. These results reveal the importance of conformational heterogeneity of active-site loops in controlling catalytic activity and directing evolutionary trajectories.

A Test for the Rapid Detection of the Cefazolin Inoculum Effect in Methicillin-Susceptible Staphylococcus aureus.

The cefazolin inoculum effect (CzIE) has been associated with therapeutic failures and mortality in invasive methicillin-susceptible Staphylococcus aureus (MSSA) infections. A diagnostic test to detect the CzIE is not currently available. We developed a rapid (∼3 h) CzIE colorimetric test to detect staphylococcal-ß-lactamase (BlaZ) activity in supernatants after ampicillin induction. The test was validated using 689 bloodstream MSSA isolates recovered from Latin America and the United States. The cefazolin MIC determination at a high inoculum (107 CFU/ml) was used as a reference standard (cutoff ≥16 µg/ml). All isolates underwent genome sequencing. A total of 257 (37.3%) of MSSA isolates exhibited the CzIE by the reference standard method. The overall sensitivity and specificity of the colorimetric test was 82.5% and 88.9%, respectively. Sensitivity in MSSA isolates harboring type A BlaZ (the most efficient enzyme against cefazolin) was 92.7% with a specificity of 87.8%. The performance of the test was lower against type B and C enzymes (sensitivities of 53.3% and 72.3%, respectively). When the reference value was set to ≥32 µg/ml, the sensitivity for isolates carrying type A enzymes was 98.2%. Specificity was 100% for MSSA lacking blaZ The overall negative predictive value ranged from 81.4% to 95.6% in Latin American countries using published prevalence rates of the CzIE. MSSA isolates from the United States were genetically diverse, with no distinguishing genomic differences from Latin American MSSA, distributed among 18 sequence types. A novel test can readily identify most MSSA isolates exhibiting the CzIE, particularly those carrying type A BlaZ. In contrast to the MIC determination using high inoculum, the rapid test is inexpensive, feasible, and easy to perform. After minor validation steps, it could be incorporated into the routine clinical laboratory workflow.

Synergistic effects of functionally distinct substitutions in ß-lactamase variants shed light on the evolution of bacterial drug resistance.

The CTX-M ß-lactamases have emerged as the most widespread extended-spectrum ß-lactamases (ESBLs) in Gram-negative bacteria. These enzymes rapidly hydrolyze cefotaxime, but not the related cephalosporin, ceftazidime. ESBL variants have evolved, however, that provide enhanced ceftazidime resistance. We show here that a natural variant at a nonactive site, i.e. second-shell residue N106S, enhances enzyme stability but reduces catalytic efficiency for cefotaxime and ceftazidime and decreases resistance levels. However, when the N106S variant was combined with an active-site variant, D240G, that enhances enzyme catalytic efficiency, but decreases stability, the resultant double mutant exhibited higher resistance levels than predicted on the basis of the phenotypes of each variant. We found that this epistasis is due to compensatory effects, whereby increased stability provided by N106S overrides its cost of decreased catalytic activity. X-ray structures of the variant enzymes in complex with cefotaxime revealed conformational changes in the active-site loop spanning residues 103-106 that were caused by the N106S substitution and relieve steric strain to stabilize the enzyme, but also alter contacts with cefotaxime and thereby reduce catalytic activity. We noted that the 103-106 loop conformation in the N106S-containing variants is different from that of WT CTX-M but nearly identical to that of the non-ESBL, TEM-1 ß-lactamase, having a serine at the 106 position. Therefore, residue 106 may serve as a "switch" that toggles the conformations of the 103-106 loop. When it is serine, the loop is in the non-ESBL, TEM-like conformation, and when it is asparagine, the loop is in a CTX-M-like, cefotaximase-favorable conformation.

WDR26 functions as a scaffolding protein to promote Gßγ-mediated phospholipase C ß2 (PLCß2) activation in leukocytes.

We have recently identified WDR26 as a novel WD40 repeat protein that binds Gßγ and promotes Gßγ signaling during leukocyte migration. Here, we have determined the mechanism by which WDR26 enhances Gßγ-mediated phospholipase C ß2 (PLCß2) activation in leukocytes. We show that WDR26 not only directly bound Gßγ but also PLCß2. The binding sites of WDR26 and PLCß2 on Gß1γ2 were overlapping but not identical. WDR26 used the same domains for binding Gßγ and PLCß but still formed a signaling complex with Gßγ and PLCß2 probably due to the fact that WDR26 formed a higher order oligomer through its Lis homology and C-terminal to LisH (LisH-CTLH) and WD40 domains. Additional studies indicated that the formation of higher order oligomers was required for WDR26 to promote PLCß2 interaction with and activation by Gßγ. Moreover, WDR26 was required for PLCß2 translocation from the cytosol to the membrane in polarized leukocytes, and the translocation of PLCß2 was sufficient to cause partial activation of PLCß2. Collectively, our data indicate that WDR26 functions as a scaffolding protein to promote PLCß2 membrane translocation and interaction with Gßγ, thereby enhancing PLCß2 activation in leukocytes. These findings have identified a novel mechanism of regulating Gßγ signaling through a scaffolding protein.

Cerecidins, novel lantibiotics from Bacillus cereus with potent antimicrobial activity.

Lantibiotics are ribosomally synthesized and posttranslationally modified antimicrobial peptides that are widely produced by Gram-positive bacteria, including many species of the Bacillus group. In the present study, one novel gene cluster coding lantibiotic cerecidins was unveiled in Bacillus cereus strain As 1.1846 through genomic mining and PCR screening. The designated cer locus is different from that of conventional class II lantibiotics in that it included seven tandem precursor cerA genes, one modification gene (cerM), two processing genes (cerT and cerP), one orphan regulator gene (cerR), and two immunity genes (cerF and cerE). In addition, one unprecedented quorum sensing component, comQXPA, was inserted between cerM and cerR. The expression of cerecidins was not detected in this strain of B. cereus, which might be due to repressed transcription of cerM. We constitutively coexpressed cerA genes and cerM in Escherichia coli, and purified precerecidins were proteolytically processed with the endoproteinase GluC and a truncated version of putative serine protease CerP. Thus, two natural variants of cerecidins A1 and A7 were obtained which contained two terminal nonoverlapping thioether rings rarely found in lantibiotics. Both cerecidins A1 and A7 were active against a broad spectrum of Gram-positive bacteria. Cerecidin A7, especially its mutant Dhb13A, showed remarkable efficacy against multidrug-resistant Staphylococcus aureus (MDRSA), vancomycin-resistant Enterococcus faecalis (VRE), and even Streptomyces.

The WD40 repeat protein WDR26 binds Gßγ and promotes Gßγ-dependent signal transduction and leukocyte migration.

The Gßγ subunits of heterotrimeric G proteins transmit signals to control many cellular processes, including leukocyte migration. Gßγ signaling may regulate and be regulated by numerous signaling partners. Here, we reveal that WDR26, a member of the WD40 repeat protein family, directly bound free Gßγ in vitro, and formed a complex with endogenous Gßγ in Jurkat T cells stimulated by the chemokine SDF1α. Suppression of WDR26 by siRNAs selectively inhibited Gßγ-dependent phospholipase Cß and PI3K activation, and attenuated chemotaxis in Jurkat T cells and differentiated HL60 cells in vitro and Jurkat T cell homing to lymphoid tissues in scid mice. Similarly, disruption of the WDR26/Gßγ interaction via expression of a WDR26 deletion mutant impaired Gßγ signaling and Jurkat T cell migration, indicating that the function of WDR26 depends on its binding to Gßγ. Additional data show that WDR26 also controlled RACK1, a negative regulator, in binding Gßγ and inhibiting leukocyte migration. Collectively, these experiments identify WDR26 as a novel Gßγ-binding protein that is required for the efficacy of Gßγ signaling and leukocyte migration.

A critical role of Gbetagamma in tumorigenesis and metastasis of breast cancer.

A growing body of evidence indicates that G protein-coupled receptors (GPCRs) are involved in breast tumor progression and that targeting GPCRs may be a novel adjuvant strategy in cancer treatment. However, due to the redundant role of multiple GPCRs in tumor development, it may be necessary to target a common signaling component downstream of these receptors to achieve maximum efficacy. GPCRs transmit signals through heterotrimeric G proteins composed of Gα and Gßγ subunits. Here we evaluated the role of Gßγ in breast tumor growth and metastasis both in vitro and in vivo. Our data show that blocking Gßγ signaling with Gα(t) or small molecule inhibitors blocked serum-induced breast tumor cell proliferation as well as tumor cell migration induced by various GPCRs in vitro. Moreover, induced expression of Gα(t) in MDA-MB-231 cells inhibited primary tumor formation and retarded growth of existing breast tumors in nude mice. Blocking Gßγ signaling also dramatically reduced the incidence of spontaneous lung metastasis from primary tumors and decreased tumor formation in the experimental lung metastasis model. Additional studies indicate that Gßγ signaling may also play a role in the generation of a tumor microenvironment permissive for tumor progression, because the inhibition of Gßγ signaling attenuated leukocyte infiltration and angiogenesis in primary breast tumors. Taken together, our data demonstrate a critical role of Gßγ signaling in promoting breast tumor growth and metastasis and suggest that targeting Gßγ may represent a novel therapeutic approach for breast cancer.

Deep Mutational Scanning Reveals the Active-Site Sequence Requirements for the Colistin Antibiotic Resistance Enzyme MCR-1.

Colistin (polymyxin E) and polymyxin B have been used as last-resort agents for treating infections caused by multidrug-resistant Gram-negative bacteria. However, their efficacy has been challenged by the emergence of the mobile colistin resistance gene mcr-1, which encodes a transmembrane phosphoethanolamine (PEA) transferase enzyme, MCR-1. The enzyme catalyzes the transfer of the cationic PEA moiety of phosphatidylethanolamine (PE) to lipid A, thereby neutralizing the negative charge of lipid A and blocking the binding of positively charged polymyxins. This study aims to facilitate understanding of the mechanism of the MCR-1 enzyme by investigating its active-site sequence requirements. For this purpose, 23 active-site residues of MCR-1 protein were randomized by constructing single-codon randomization libraries. The libraries were individually selected for supporting Escherichia coli cell growth in the presence of colistin or polymyxin B. Deep sequencing of the polymyxin-resistant clones revealed that wild-type residues predominates at 17 active-site residue positions, indicating these residues play critical roles in MCR-1 function. These residues include Zn2+-chelating residues as well as residues that may form a hydrogen bond network with the PEA moiety or make hydrophobic interactions with the acyl chains of PE. Any mutations at these residues significantly decrease polymyxin resistance levels and the PEA transferase activity of the MCR-1 enzyme. Therefore, deep sequencing of the randomization libraries of MCR-1 enzyme identifies active-site residues that are essential for its polymyxin resistance function. Thus, these residues may be utilized as targets to develop inhibitors to circumvent MCR-1-mediated polymyxin resistance. IMPORTANCE Polymyxin antibiotics are used as last-line antibiotics in treating infections caused by multidrug-resistant pathogens. However, widespread use of polymyxins has led to the emergence of resistance. Although multiple mechanisms for resistance exist, that due to mcr-1 is a particular concern, as it can be readily transferred among bacterial pathogens. The mcr-1 gene encodes a transmembrane phosphoethanolamine (PEA) transferase that modifies lipid A to block the binding of polymyxin antibiotics. We utilized random mutagenesis coupled with next-generation sequencing to determine the amino acid sequence requirements of 23 residues in and near the active site of MCR-1. We show that the enzyme has stringent sequence requirements, with 75% of the residues examined being essential for function. Coupled with the finding that these residues are largely conserved among PEA enzymes, the results suggest inhibitors that bind near these sites will broadly inhibit MCR-1 and other enzymes of this class.

[Advances in the study of nisin resistance--a review].

Nisin, a lantibiotic produced by some species of Lactococcus lactis, has broad antibacterial spectrum against Gram-positive bacteria species, especially those with close phylogenetic relationship to the nisin producing strain. The broad use of nisin did not lead to widespread resistance. However, some non-nisin producing bacteria could develop certain mechanisms of resistance against nisin when growing under laboratory or nature selection pressure. Nisin resistance mainly involved two strategies, namely, the non-specific physiological isolation mechanism (by the change of cell wall or membrane structure and composition) and the specific protease-mediated mechanism. This review introduced the advances in the study of nisin resistance mechanism.

Streptococcus pyogenes genes that promote pharyngitis in primates.

Streptococcus pyogenes (group A streptococcus; GAS) causes 600 million cases of pharyngitis annually worldwide. There is no licensed human GAS vaccine despite a century of research. Although the human oropharynx is the primary site of GAS infection, the pathogenic genes and molecular processes used to colonize, cause disease, and persist in the upper respiratory tract are poorly understood. Using dense transposon mutant libraries made with serotype M1 and M28 GAS strains and transposon-directed insertion sequencing, we performed genome-wide screens in the nonhuman primate (NHP) oropharynx. We identified many potentially novel GAS fitness genes, including a common set of 115 genes that contribute to fitness in both genetically distinct GAS strains during experimental NHP pharyngitis. Targeted deletion of 4 identified fitness genes/operons confirmed that our newly identified targets are critical for GAS virulence during experimental pharyngitis. Our screens discovered many surface-exposed or secreted proteins - substrates for vaccine research - that potentially contribute to GAS pharyngitis, including lipoprotein HitA. Pooled human immune globulin reacted with purified HitA, suggesting that humans produce antibodies against this lipoprotein. Our findings provide new information about GAS fitness in the upper respiratory tract that may assist in translational research, including developing novel vaccines.

Novel mechanism for nisin resistance via proteolytic degradation of nisin by the nisin resistance protein NSR.

Nisin is a 34-residue antibacterial peptide produced by Lactococcus lactis that is active against a wide range of gram-positive bacteria. In non-nisin-producing L. lactis, nisin resistance could be conferred by a specific nisin resistance gene (nsr), which encodes a 35-kDa nisin resistance protein (NSR). However, the mechanism underlying NSR-mediated nisin resistance is poorly understood. Here we demonstrated that the protein without the predicted N-terminal signal peptide sequence, i.e., NSRSD, could proteolytically inactivate nisin in vitro by removing six amino acids from the carboxyl "tail" of nisin. The truncated nisin (nisin(1-28)) displayed a markedly reduced affinity for the cell membrane and showed significantly diminished pore-forming potency in the membrane. A 100-fold reduction of bactericidal activity was detected for nisin(1-28) in comparison to that for the intact nisin. In vivo analysis indicated that NSR localized on the cell membrane and endowed host strains with nisin resistance by degrading nisin as NSRSD did in vitro, whereas NSRSD failed to confer resistance upon the host strain. In conclusion, we showed that NSR is a nisin-degrading protease. This NSR-mediated proteolytic cleavage represents a novel mechanism for nisin resistance in non-nisin-producing L. lactis.

Extensively Drug-Resistant Pseudomonas aeruginosa ST309 Harboring Tandem Guiana Extended Spectrum ß-Lactamase Enzymes: A Newly Emerging Threat in the United States.

BACKGROUND: Treatment of serious infections due to multidrug-resistant (MDR) Pseudomonas aeruginosa remains a challenge, despite the introduction of novel therapeutics. In this study, we report 2 extensively drug-resistant clinical isolates of sequence type (ST) 309 P aeruginosa resistant to all ß-lactams, including the novel combinations ceftolozane/tazobactam, ceftazidime/avibactam, and meropenem/vaborbactam. METHODS: Isolates were sequenced using both short-read (Illumina) and long-read technology to identify resistance determinants, polymorphisms (compared with P aeruginosa PAO1), and reconstruct a phylogenetic tree. A pair of ß-lactamases, Guiana extended spectrum ß-lactamase (GES)-19 and GES-26, were cloned and expressed in a laboratory strain of Escherichia coli to examine their relative impact on resistance. Using cell lysates from E coli expressing the GES genes individually and in tandem, we determined relative rates of hydrolysis for nitrocefin and ceftazidime. RESULTS: Two ST309 P aeruginosa clinical isolates were found to harbor the extended spectrum ß-lactamases GES-19 and GES-26 clustered in tandem on a chromosomal class 1 integron. The presence of both enzymes in E coli was associated with significantly elevated minimum inhibitory concentrations to aztreonam, cefepime, meropenem, ceftazidime/avibactam, and ceftolozane/tazobactam, compared with those expressed individually. The combination of ceftazidime/avibactam plus aztreonam was active in vitro and used to achieve cure in one patient. Phylogenetic analysis revealed ST309 P aeruginosa are closely related to MDR strains from Mexico also carrying tandem GES. CONCLUSIONS: The presence of tandem GES-19 and GES-26 is associated with resistance to all ß-lactams, including ceftolozane/tazobactam. Phylogenetic analysis suggests that ST309 P aeruginosa may be an emerging threat in the United States.

Differential active site requirements for NDM-1 ß-lactamase hydrolysis of carbapenem versus penicillin and cephalosporin antibiotics.

New Delhi metallo-ß-lactamase-1 exhibits a broad substrate profile for hydrolysis of the penicillin, cephalosporin and 'last resort' carbapenems, and thus confers bacterial resistance to nearly all ß-lactam antibiotics. Here we address whether the high catalytic efficiency for hydrolysis of these diverse substrates is reflected by similar sequence and structural requirements for catalysis, i.e., whether the same catalytic machinery is used to achieve hydrolysis of each class. Deep sequencing of randomized single codon mutation libraries that were selected for resistance to representative antibiotics reveal stringent sequence requirements for carbapenem versus penicillin or cephalosporin hydrolysis. Further, the residue positions required for hydrolysis of penicillins and cephalosporins are a subset of those required for carbapenem hydrolysis. Thus, while a common core of residues is used for catalysis of all substrates, carbapenem hydrolysis requires an additional set of residues to achieve catalytic efficiency comparable to that for penicillins and cephalosporins.

Upregulated WDR26 serves as a scaffold to coordinate PI3K/ AKT pathway-driven breast cancer cell growth, migration, and invasion.

The phosphatidylinositol 3-kinase (PI3K)/AKT pathway transmits signals downstream of receptor tyrosine kinases and G protein-coupled receptors (GPCRs), and is one of the most dysregulated pathways in breast cancer. PI3Ks and AKTs consist of multiple isoforms that play distinct and even opposite roles in breast cancer cell growth and metastasis. However, it remains unknown how the activities of various PI3K and AKT isoforms are coordinated during breast cancer progression. Previously, we showed WDR26 is a novel WD40 protein that binds Gßγ and promotes Gßγ signaling. Here, we demonstrate that WDR26 is overexpressed in highly malignant breast tumor cell lines and human breast cancer samples, and that WDR26 overexpression correlates with shortened survival of breast cancer patients. In highly malignant cell lines (MDA-MB231, DU4475 and BT549), downregulation of WDR26 expression selectively alleviated GPCR- but not EGF receptor-stimulated PI3K/AKT signaling and tumor cell growth, migration and invasion. In contrast, in a less malignant cell line (MCF7), WDR26 overexpression had the opposite effect. Additional studies indicate that downstream of GPCR stimulation, WDR26 serves as a scaffold that fosters assembly of a specific signaling complex consisting of Gßγ, PI3Kß and AKT2. In an orthotopic xenograft mouse model of breast cancer, disrupting formation of this complex, by overexpressing WDR26 mutants in MDA-MB231 cells, abrogated PI3K/AKT activation and tumor cell growth and metastasis. Together, our results identify a novel mechanism regulating GPCR-dependent activation of the PI3K/AKT signaling axis in breast tumor cells, and pinpoint WDR26 as a potential therapeutic target for breast cancer.

Deep Sequencing of Random Mutant Libraries Reveals the Active Site of the Narrow Specificity CphA Metallo-ß-Lactamase is Fragile to Mutations.

CphA is a Zn(2+)-dependent metallo-ß-lactamase that efficiently hydrolyzes only carbapenem antibiotics. To understand the sequence requirements for CphA function, single codon random mutant libraries were constructed for residues in and near the active site and mutants were selected for E. coli growth on increasing concentrations of imipenem, a carbapenem antibiotic. At high concentrations of imipenem that select for phenotypically wild-type mutants, the active-site residues exhibit stringent sequence requirements in that nearly all residues in positions that contact zinc, the substrate, or the catalytic water do not tolerate amino acid substitutions. In addition, at high imipenem concentrations a number of residues that do not directly contact zinc or substrate are also essential and do not tolerate substitutions. Biochemical analysis confirmed that amino acid substitutions at essential positions decreased the stability or catalytic activity of the CphA enzyme. Therefore, the CphA active - site is fragile to substitutions, suggesting active-site residues are optimized for imipenem hydrolysis. These results also suggest that resistance to inhibitors targeted to the CphA active site would be slow to develop because of the strong sequence constraints on function.

Ablation of the GNB3 gene in mice does not affect body weight, metabolism or blood pressure, but causes bradycardia.

G protein ß3 (Gß3) is an isoform of heterotrimeric G protein ß subunits involved in transducing G protein coupled receptor (GPCR) signaling. Polymorphisms in Gß3 (GNB3) are associated with many human disorders (e.g. hypertension, diabetes and obesity) but the role of GNB3 in these pathogeneses remains unclear. Here, Gß3-null mice (GNB3(-/-)) were characterized to determine how Gß3 functions to regulate blood pressure, body weight and metabolism. We found Gß3 expression restricted to limited types of tissues, including the retina, several regions of the brain and heart ventricles. Gß3-deficient mice were normal as judged by body weight gain by age or by feeding with high-fat diet (HFD); glucose tolerance and insulin sensitivity; baseline blood pressure and angiotensin II infusion-induced hypertension. During tail-cuff blood pressure measurements, however, Gß3-null mice had slower heart rates (~450 vs ~500 beats/min). This bradycardia was not observed in isolated and perfused Gß3-null mouse hearts. Moreover, mouse hearts isolated from GNB3(-/-) and controls responded equivalently to muscarinic receptor- and ß-adrenergic receptor-stimulated bradycardia and tachycardia, respectively. Since no difference was seen in isolated hearts, Gß3 is unlikely to be involved directly in the GPCR signaling activity that controls heart pacemaker activity. These results demonstrate that although Gß3 appears dispensable in mice for the regulation of blood pressure, body weight and metabolic features associated with obesity and diabetes, Gß3 may regulate heart rate.

Bovicin HJ50-like lantibiotics, a novel subgroup of lantibiotics featured by an indispensable disulfide bridge.

Lantibiotics are ribosomally-synthesized and posttranslationally modified peptides with potent antimicrobial activities. Discovery of novel lantibiotics has been greatly accelerated with the soaring release of genomic information of microorganisms. As a unique class II lantibiotic, bovicin HJ50 is produced by Streptococcus bovis HJ50 and contains one rare disulfide bridge. By using its precursor BovA as a drive sequence, 16 BovA-like peptides were revealed in a wide variety of species. From them, three representative novel lan loci from Clostridium perfringens D str. JGS1721, Bacillus cereus As 1.348 and B. thuringiensis As 1.013 were identified by PCR screening. The corresponding mature lantibiotics designated perecin, cerecin and thuricin were obtained and structurally elucidated to be bovicin HJ50-like lantibiotics especially by containing a conserved disulfide bridge. The disulfide bridge was substantiated to be essential for the function of bovicin HJ50-like lantibiotics as its disruption eliminated their antimicrobial activities. Further analysis indicated that the disulfide bridge played a crucial role in maintaining the hydrophobicity of bovicin HJ50, which might facilitate it to exert antimicrobial function. This study unveiled a novel subgroup of disulfide-containing lantibiotics from bacteria of different niches and further demonstrated the indispensable role of disulfide bridge in these novel bovicin HJ50-like lantibiotics.

The Gß3 splice variant associated with the C825T gene polymorphism is an unstable and functionally inactive protein.

A splice variant of Gß3, termed Gß3s, has been associated with the C825T polymorphism in the Gß3 gene and linked with many human disorders. However, the biochemical properties and functionality of Gß3s remain controversial. Here, using multidisciplinary approaches including co-immunoprecipitation analysis and bioluminescence resonance energy transfer (BRET) measurements, we showed that unlike Gß3, Gß3s failed to form complexes with either Gγ or Gα subunits. Moreover, using a mutant Gγ2 deficient in lipid modification to purify Gß3s from Sf9 cells without the use of detergents, we further showed that the failure of Gß3s to form dimers with Gγ was not due to the instability of the dimers in detergents, but rather, reflected the intrinsic properties of Gß3s. Additional studies indicated that Gß3s is unstable, and unable to localize properly to the plasma membrane and to activate diverse Gßγ effectors including PLCß2/3, PI3Kγ, ERKs and the Rho guanine exchange factor (RhoGEF) PLEKHG2. Thus, these data suggest that the pathological effects of Gß3 C825T polymorphism may result from the downregulation of Gß3 function. However, we found that the chemokine SDF1α transmits signals primarily through Gß1 and Gß2, but not Gß3, to regulate chemotaxis of several human lymphocytic cell lines, indicating the effects of Gß3 C825T polymorphism are likely to be tissue and/or stimuli specific and its association with various disorders in different tissues should be interpreted with great caution.