P-type ATPase zinc transporter Rv3270 of Mycobacterium tuberculosis enhances multi-drug efflux activity.

Metal homeostasis is maintained by the uptake, storage and efflux of metal ions that are necessary for the survival of the bacterium. Homeostasis is mostly regulated by a group of transporters categorized as ABC transporters and P-type ATPases. On the other hand, efflux pumps often play a role in drug-metal cross-resistance. Here, with the help of antibiotic sensitivity, antibiotic/dye accumulation and semi-quantitative biofilm formation assessments we report the ability of Rv3270, a P-type ATPase known for its role in combating Mn2+ and Zn2+ metal ion toxicity in Mycobacterium tuberculosis, in influencing the extrusion of multiple structurally unrelated drugs and enhancing the biofilm formation of Escherichia coli and Mycobacterium smegmatis. Overexpression of Rv3270 increased the tolerance of host cells to norfloxacin, ofloxacin, sparfloxacin, ampicillin, oxacillin, amikacin and isoniazid. A significantly lower accumulation of norfloxacin, ethidium bromide, bocillin FL and levofloxacin in cells harbouring Rv3270 as compared to host cells indicated its role in enhancing efflux activity. Although over-expression of Rv3270 did not alter the susceptibility levels of levofloxacin, rifampicin and apramycin, the presence of a sub-inhibitory concentration of Zn2+ resulted in low-level tolerance towards these drugs. Of note, the expression of Rv3270 enhanced the biofilm-forming ability of the host cells strengthening its role in antimicrobial resistance. Therefore, the study indicated that the over-expression of Rv3270 enhances the drug efflux activity of the micro-organism where zinc might facilitate drug-metal cross-resistance for some antibiotics.

The MSMEG_1586 of M. smegmatis Is a Penicillin-Interactive Enzyme That Can Potentially Hydrolyse Aztreonam and Cephalosporins.

Mycobacteria are intrinsically resistant to beta-lactams as they possess several putative penicillin-interactive enzymes (PIEs), some of those are with dual-activity, namely DD-carboxypeptidase and beta-lactamase. Here, with help of molecular approaches, we elucidated the nature of one such putative PIE, MSMEG_1586, in Mycobacterium smegmatis. The in vivo expression of the membrane-bound form of MSMEG_1586 enhanced the beta-lactam resistance of a beta-lactamase deleted host E. coli strain (AM1OC), particularly for aztreonam (eight-fold) and cephalosporins (8-16 fold). To understand the reason for such elevation of resistance, soluble-form of MSMEG_1586 (sMSMEG_1586) was created by removing signal peptides and partially eliminating the amphipathic helix, and finally, expressed and purified. The purified sMSMEG_1586 was active and manifested a strong penicillin-binding affinity as shown by its ability to bind to fluorescent penicillin (Bocillin-FL). Interestingly, the steady-state kinetics apparently confirmed the hydrolytic ability of sMSMEG_1586 towards cefotaxime and aztreonam where hydrolysing aztreonam is a unique and rare behaviour among the beta-lactamases. However, sMSMEG_1586 was devoid of exerting DD-carboxypeptidase like activity. Finally, in silico analysis of MSMEG_1586 revealed a special folding that resembles class C beta-lactamase, except for the absence of a characteristic R2 loop. Overall, MSMEG_1586 could be categorized as a cephalosporinase with the ability to hydrolyse aztreonam.

Antimicrobial Peptides Designed against the Ω-Loop of Class A ß-Lactamases to Potentiate the Efficacy of ß-Lactam Antibiotics.

Class A serine ß-lactamases (SBLs) have a conserved non-active site structural domain called the omega loop (Ω-loop), in which a glutamic acid residue is believed to be directly involved in the hydrolysis of ß-lactam antibiotics by providing a water molecule during catalysis. We aimed to design and characterise potential pentapeptides to mask the function of the Ω-loop of ß-lactamases and reduce their efficacy, along with potentiating the ß-lactam antibiotics and eventually decreasing ß-lactam resistance. Considering the Ω-loop sequence as a template, a group of pentapeptide models were designed, validated through docking, and synthesised using solid-phase peptide synthesis (SPPS). To check whether the ß-lactamases (BLAs) were inhibited, we expressed specific BLAs (TEM-1 and SHV-14) and evaluated the trans-expression through a broth dilution method and an agar dilution method (HT-SPOTi). To further support our claim, we conducted a kinetic analysis of BLAs with the peptides and employed molecular dynamics (MD) simulations of peptides. The individual presence of six histidine-based peptides (TSHLH, ETHIH, ESRLH, ESHIH, ESRIH, and TYHLH) reduced ß-lactam resistance in the strains harbouring BLAs. Subsequently, we found that the combinational effect of these peptides and ß-lactams sensitised the bacteria towards the ß-lactam drugs. We hypothesize that the antimicrobial peptides obtained might be considered among the novel inhibitors that can be used specifically against the Ω-loop of the ß-lactamases.

A NiCoT family metal transporter of Mycobacterium tuberculosis (Rv2856/NicT) behaves as a drug efflux pump that facilitates cross-resistance to antibiotics.

Metals often act as a facilitator in the proliferation and persistence of antibiotic resistance. Efflux pumps play key roles in the co-selection of metal and antibiotic resistance. Here, we report the ability of a putative nickel/cobalt transporter (NiCoT family), Rv2856 or NicT of Mycobacterium tuberculosis (Mtb), to transport metal and antibiotics and identified some key amino acid residues that are important for its function. Ectopic expression of NicT in Escherichia coli CS109 resulted in the increase of intracellular nickel uptake. Additionally, enhanced tolerance towards several antibiotics (norfloxacin, sparfloxacin, ofloxacin, gentamicin, nalidixic acid and isoniazid) was observed with NicT overexpression in E. coli and Mycobacterium smegmatis. A comparatively lower intracellular accumulation of norfloxacin upon NicT overexpression than that of the cells without NicT indicated the involvement of NicT in an active efflux process. Although expression of NicT did not alter the sensitivity towards kanamycin, doxycycline, tetracycline, apramycin, neomycin and ethambutol, the presence of a sub-inhibitory dose of Ni2+ resulted in the manifestation of low-level tolerance towards these drugs. Further, substitution of four residues (H77I, D82I, H83L and D227I) in the conserved regions of NicT by isoleucine and leucine resulted in reduced to nearly complete loss of the transport function for both metals and antimicrobials. Therefore, the study suggests that nickel transporter Rv2856/NicT may actively export different drugs and the presence of nickel might drive the cross-resistance to some of the antibiotics.

Deciphering the role of residues in the loops nearing the active site of OXA-58 in imparting beta-lactamase activity.

The existence of OXA-58 carbapenemase alone or in combination with other beta-lactam resistance factors poses significant beta-lactam resistance. The exact mechanism of action of OXA type beta-lactamases is debatable due to the involvement of multiple residues within or outside the active site. In the present work, we have elucidated the relative role of residues present in the putative omega (W169, L170, K171) and ß6-ß7 (A226 and D228) loops on the activity of OXA-58 by substituting into alanine (and aspartate for A226) through site-directed mutagenesis. E. coli cells harbouring OXA-58, substituted at the putative omega loop, manifest a significant decrease in the beta-lactam resistance profile than that of the cells expressing OXA-58. Further, a reduction in the catalytic efficiency is observed for the purified variants of OXA-58 carrying individual substitutions in the putative omega loop than that of OXA-58. However, the addition of NaHCO3 (for carbamylation of K86) increases catalytic efficiency of the individual protein as revealed by nitrocefin hydrolysis assay and steady state kinetics. Moreover, W169A and K171A substitutions show significant effects on the thermal stability of OXA-58. Therefore, we conclude that the putative omega loop residues W169, L170 and K171, individually, have significant role in the activity and stability of OXA-58, mostly by stabilising carbamylated lysine of active site.

Comparative insight into the roles of the non active-site residues E169 and N173 in imparting the beta-lactamase activity of CTX-M-15.

CTX-M-15 is a major extended-spectrum beta-lactamase disseminated throughout the globe. The roles of amino acids present in the active-site are widely studied though little is known about the role of the amino acids lying at the close proximity of the CTX-M-15 active-site. Here, by using site-directed mutagenesis we attempted to decipher the role of individual amino acids lying outside the active-site in imparting the beta-lactamase activity of CTX-M-15. Based on the earlier evidence, three amino acid residues namely, Glu169, Asp173 and Arg277 were substituted with alanine. The antibiotic susceptibility of E. coli cells harboring E169A and N173A substituted CTX-M-15 were enhanced by ∼ >32 fold for penicillins and ∼ 4-32 fold for cephalosporins, in comparison to CTX-M-15. However, cells carrying CTX-M-15_R277A did not show a significant difference in antibiotic susceptibility as compared to the wild-type. Further, the catalytic efficiency of the purified CTX-M-15_E169A and CTX-M-15_N173A were compromised when compared with the efficient beta-lactam hydrolysis of purified CTX-M-15. Moreover, the thermal stability of the mutated proteins CTX-M-15_E169A and CTX-M-15_N173A were reduced as compared to the wild type CTX-M-15. Therefore, we conclude that E169 and N173 are crucial non-active-site amino acids that are able to govern the CTX-M-15 activity.

PBP4 and PBP5 are involved in regulating exopolysaccharide synthesis during Escherichia coli biofilm formation.

Escherichia coli low-molecular-mass (LMM) Penicillin-binding proteins (PBPs) help in hydrolysing the peptidoglycan fragments from their cell wall and recycling them back into the growing peptidoglycan matrix, in addition to their reported involvement in biofilm formation. Biofilms are external slime layers of extra-polymeric substances that sessile bacterial cells secrete to form a habitable niche for themselves. Here, we hypothesize the involvement of Escherichia coli LMM PBPs in regulating the nature of exopolysaccharides (EPS) prevailing in its extra-polymeric substances during biofilm formation. Therefore, this study includes the assessment of physiological characteristics of E. coli CS109 LMM PBP deletion mutants to address biofilm formation abilities, viability and surface adhesion. Finally, EPS from parent CS109 and its ΔPBP4 and ΔPBP5 mutants were purified and analysed for sugars present. Deletions of LMM PBP reduced biofilm formation, bacterial adhesion and their viability in biofilms. Deletions also diminished EPS production by ΔPBP4 and ΔPBP5 mutants, purification of which suggested an increased overall negative charge compared with their parent. Also, EPS analyses from both mutants revealed the appearance of an unusual sugar, xylose, that was absent in CS109. Accordingly, the reason for reduced biofilm formation in LMM PBP mutants may be speculated as the subsequent production of xylitol and a hindrance in the standard flow of the pentose phosphate pathway.

MSMEG_2432 of Mycobacterium smegmatis mc2155 is a dual function enzyme that exhibits DD-carboxypeptidase and ß-lactamase activities.

Mycobacterial peptidoglycan (PG) is an unsolved puzzle due to its complex structure and involvement of multiple enzymes in the process of its remodelling. dd-Carboxypeptidases are low molecular mass penicillin-binding proteins (LMM-PBPs) that catalyzes the cleavage of terminal d-Ala of muramyl pentapeptide branches and thereby helps in the PG remodelling process. Here, we have assigned the function of a putative LMM-PBP, MSMEG_2432 of Mycobacterium smegmatis, by showing that it exhibits both dd-CPase and ß-lactamase activities. Like conventional dd-CPase (PBP5 from E. coli), upon ectopic complementation in a deformed seven PBP deletion mutant of E. coli, MSMEG_2432 has manifested its ability to restore ~75 % of the cell population to their normal rod shape. Further, in vitrodd-CPase assay has confirmed its ability to release terminal d-Ala from the synthetic tripeptide and the peptidoglycan mimetic pentapeptide substrates ending with d-Ala-d-Ala. Also, elevated resistance against penicillins and cephalosporins upon ectopic expression of MSMEG_2432 suggests the presence of ß-lactamase activity, which is further confirmed in vitro through nitrocefin hydrolysis assay. Moreover, it is found apparent that D169A substitution in MSMEG_2432 influences both of its in vivo and in vitrodd-CPase and ß-lactamase activities. Thus, we infer that MSMEG_2432 is a dual function enzyme that possesses both dd-CPase and ß-lactamase activities.

Glutamic acid at position 152 and serine at position 191 are key residues required for the metallo-ß-lactamase activity of NDM-7.

New Delhi metallo-ß-lactamase (NDM) is of significant public-health concern due to its enormous potential to hydrolyse all of the major ß-lactams, including carbapenems. Previous reports indicate that amino acid substitutions affect NDM activity despite being located outside of the active site. In this study, we attempted to identify specific mutations in loops near the active site that can influence the function of NDM-7. Overall, six substitutions were performed through site-directed mutagenesis near the active-site of NDM-7 and the change in antimicrobial resistance was subsequently monitored by expressing each mutant in a suitable bacterial host. Among the six mutants, serine at position 191 (S191) and glutamic acid at position 152 (E152) were identified as the most influencing residues for NDM-7. ß-Lactam resistance of NDM-7 was remarkably affected by substitution of both residues with alanine, and the results were in accordance with the changes in kinetic parameters. Purified NDM-7 ordinarily hydrolyses ß-lactams efficiently, but purified NDM-7_E152A, NDM-7_S191A and the double mutant NDM-7_E152A+S191A had lost their ability to hydrolyse the ß-lactams tested, especially penicillins and carbapenems. Although the substitutions did not affect the overall folding pattern of the NDM-7 enzyme, substantial differences in thermal stability were observed. Therefore, we hypothesise that the residues S191 and E152 together play a crucial role in conferring the ß-lactamase character of NDM-7.

Rapid Fluorescent-Based Detection of New Delhi Metallo-ß-Lactamases by Photo-Cross-Linking Using Conjugates of Azidonaphthalimide and Zinc(II)-Chelating Motifs.

A method for rapid detection of metallo-ß-lactamases NDM-5 and NDM-7 using conjugates of azidonaphthalimide and Zn(II) chelating motifs (like sulfonamides, hydroxamate, and terpyridine) is described. Incubation and irradiation, followed by gel electrophoresis, clearly show the presence of NDMs. The o-sulfonamide-based probe has the highest efficiency of detection for both the NDMs. The proteins are detectable at nM concentrations, and the method is also selective, works both in vitro and in vivo, as revealed by cellular imaging and also with clinical isolates.

Bio-surfactin stabilised silver nanoparticles exert inhibitory effect over New-Delhi metallo-beta-lactamases (NDMs) and the cells harbouring NDMs.

Silver nanoparticles (AgNPs) are used as an antimicrobial agent since the ages. However, it is unknown whether AgNPs exert inhibitory effects over the bacterial cells carrying metallo-beta-lactamases (MBLs). Here, using bio-surfactin stabilised AgNPs having a size range from 5 to 25 nm we established its antimicrobial effects against NDMs harbouring cells. Antimicrobial effectiveness of AgNPs is assessed on the E. coli cells carrying New Delhi MBL (NDM) genes, which shows that the cells expressing NDM becomes susceptible to AgNPs and when combined with various groups of beta-lactam a synergistic increase in sensitivity is observed. Purified NDMs are also inhibited by AgNPs as revealed by the hydrolysis of nitrocefin (a chromogenic cephalosporin), though the expression NDM genes remain unchanged. Further, the results obtained from biochemical analysis attribute that the Ag+ ions possibly bind to sulfhydryl (SH) group of cystine in NDMs to inactivate these enzymes. Nonetheless, these AgNPs has the ability to exert antimicrobial activity without affecting the host cell viability when used at a moderate concentration. Overall, we conclude that bio-surfactin-stabilised AgNP is a good candidate to serve as an inhibitor of NDMs, either individually or in combination with beta-lactams.

Absence of the glycosyltransferase WcaJ in Klebsiella pneumoniae ATCC13883 affects biofilm formation, increases polymyxin resistance and reduces murine macrophage activation.

Multidrug-resistant Klebsiella pneumoniae has emerged as one of the deadliest opportunistic nosocomial pathogens that forms biofilm for the establishment of chronic K. pneumoniae infections. Herein, we made an attempt to identify the genes involved in biofilm formation in the strain K. pneumoniae ATCC13883. To achieve this, we constructed mini-Tn5 transposon insertion mutants and screened them for biofilm production. We observed that the biofilm formation was enhanced in the mutant where the wcaJ gene was disrupted. WcaJ is the initiating enzyme of colanic acid synthesis and loads the first sugar (glucose-1-P) on the lipid carrier undecaprenyl phosphate. The absence of this glycosyltransferase results in the absence of colanic acid, which renders a non-mucoid phenotype to the mutant. Further, to determine the effect of mucoidy on antibiotic susceptibility, we tested the sensitivity of the strains towards different groups of antibiotics. Unlike the mucoid strains, the resistance of the non-mucoid cells was greater for polymyxins, but less for quinolones. Capsular polysaccharides are known to have a protective effect against phagocytosis, therefore we assessed the role of colanic acid in virulence by conducting infection studies on murine macrophages. Surprisingly, the ΔwcaJ strain was less efficient in macrophage activation and was not readily phagocytosed. Thus, the presence of colanic acid appeared to increase the immunogenicity of K. pneumoniae. Overall, the results indicate that the presence of colanic acid increases the vulnerability of K. pneumoniae towards both polymyxins and macrophages, implying that the mucoid strains are less threatening as compared to their high biofilm forming non-mucoid counterparts.

Role of Escherichia coli endopeptidases and dd-carboxypeptidases in infection and regulation of innate immune response.

The low-molecular-mass penicillin-binding proteins, involved in peptidoglycan recycling can also produce peptidoglycan fragments capable of activating an innate immune response in host. To investigate how these proteins in Enterobacteriaceae play a role to elicit/evade innate immune responses during infections, we deleted certain endopeptidases and dd-carboxypeptidases from Escherichia coli CS109 and studied the viability of these mutants in macrophages. The ability of infected macrophages to exert oxidative killing, express surface activation markers TLR2, MHC class II and release TNFα, were assessed. Immune responses were elevated in macrophages infected with dd-carboxypeptidase mutants but reduced for endopeptidase mutants. However, the NFκB, iNOS, and TLR2 transcripts remained elevated in macrophages infected with both mutant types. Overall, we have shown, under normal conditions endopeptidases have a tendency to elicit the immune response but their effect is suppressed by the presence of dd-carboxypeptidases. Conversely, DD-carboxypeptidases, normally, tend to reduce immune responses, as their deletions enhanced the same in macrophages. Therefore, we conclude that the roles of endopeptidases and dd-carboxypeptidases are possibly counter-active in wild-type cells where either class of enzymes suppresses each other's immunogenic properties rendering overall maintenance of low immunogenicity that helps E. coli in evading the host immune responses.

PBP Isolation and DD-Carboxypeptidase Assay.

Penicillin-binding proteins (PBPs) share the namesake because of their ability to bind penicillin or any beta-lactam antibiotic. In other words, PBPs are the targets of ß-lactam antibiotics that hold nearly 60% of the global antibiotic market. These enzymes catalyze the final stages of peptidoglycan (PG) biosynthesis by acting as transglycosylases and transpeptidases. PBPs are also involved in PG remodeling by catalyzing DD-carboxypeptidase (DD-CPase) and endopeptidase reactions. Though the cross-linking abilities of PBPs are well known, the process of remodeling is still unclear, thereby drawing attention toward the DD-CPase enzymes. Here, we describe the step-by-step procedures for isolation of the bacterial cell membrane and detection of PBPs in it, followed by the purification of PBPs (DD-CPases) by both ampicillin-affinity and nickel-nitrilotriacetic acid (Ni-NTA) chromatography. The protocols to determine the enzymatic efficiency are also elucidated. The assays are aimed to determine the kinetic parameters for the interaction of the PBP with BOCILLIN, to evaluate its acylation and deacylation rates, and with its peptide substrates, to assess its DD-CPase activity.

Two dd-Carboxypeptidases from Mycobacterium smegmatis Affect Cell Surface Properties through Regulation of Peptidoglycan Cross-Linking and Glycopeptidolipids.

During the peptidoglycan (PG) maturation of mycobacteria, the glycan strands are interlinked by both 3-3 (between two meso-diaminopimelic acids [meso-DAPs]) and 4-3 cross-links (between d-Ala and meso-DAP), though there is a predominance (60 to 80%) of 3-3 cross-links. The dd-carboxypeptidases (dd-CPases) act on pentapeptides to generate tetrapeptides that are used by ld-transpeptidases as substrates to form 3-3 cross-links. Therefore, dd-CPases play a crucial role in mycobacterial PG cross-link formation. However, the physiology of dd-CPases in mycobacteria is relatively unexplored. In this study, we deleted two dd-CPase genes, msmeg_2433 and msmeg_2432, both individually and in combination, from Mycobacterium smegmatis mc2155. Though the single dd-CPase gene deletions had no significant impact on the mycobacterial physiology, many interesting functional alterations were observed in the double-deletion mutant, viz, a predominance in PG cross-link formation was shifted from 3-3 cross-links to 4-3, cell surface glycopeptidolipid (GPL) expression was reduced, and susceptibility to ß-lactams and antitubercular agents was enhanced. Moreover, the survival rate of the double mutant within murine macrophages was higher than that of the parent. Interestingly, the complementation with any one of the dd-CPase genes could restore the wild-type phenotype. In a nutshell, we infer that the altered ratio of 4-3 to 3-3 PG cross-links might have influenced the expression of surface GPLs, colony morphology, biofilm formation, drug susceptibility, and subsistence of the cells within macrophages.IMPORTANCE The glycan strands in mycobacterial peptidoglycan (PG) are interlinked by both 3-3 and 4-3 cross-links. The dd-CPases generate tetrapeptides by acting on the pentapeptides, and ld-transpeptidases use tetrapeptides as substrates to form 3-3 cross-links. In this study, we showed that simultaneous deletions of two dd-CPases alter the nature of PG cross-linking from 3-3 cross-links to 4-3 cross-links. The deletions subsequently decrease the expression of glycopeptidolipids (significant surface lipid present in many nontuberculous mycobacteria, including Mycobacterium smegmatis) and affect other physiological parameters, like cell morphology, growth rate, biofilm formation, antibiotic susceptibility, and survival within murine macrophages. Thus, unraveling the physiology of dd-CPases might help us design antimycobacterial therapeutics in the future.

Substitution of Alanine at Position 184 with Glutamic Acid in Escherichia coli PBP5 Ω-Like Loop Introduces a Moderate Cephalosporinase Activity.

Escherichia coli PBP5, a DD-carboxypeptidase (DD-CPase), helps in maintaining cell shape and intrinsic ß-lactam resistance. Though PBP5 does not have ß-lactamase activity under physiological pH, it has a common but shorter Ω-like loop resembling class A ß-lactamases. However, such Ω-like loop lacks the key glutamic acid residue that is present in ß-lactamases. It is speculated that ß-lactamases and DD-CPases might have undergone divergent evolution leading to distinct enzymes with different substrate specificities and functions indicating the versatility of the Ω-loops. Nonetheless, direct experimental evidence favoring the idea is insufficient. Here, aiming to investigate the effect of introducing a glutamic acid residue in the PBP5 Ω-like loop, we substituted A184 to E to create PBP5_A184E. Expression of PBP5_A184E in E. coli ∆PBP5 mutant elevates the ß-lactam resistance, especially for cephalosporins. However, like PBP5, PBP5_A184E has the ability to complement the aberrantly shaped E. coli septuple PBP mutant indicating an unaffected in vivo DD-CPase activity. Biochemical and bioinformatics analyses have substantiated the dual enzyme nature of the mutated enzyme possessing both DD-CPase and ß-lactamase activities. Therefore, substitution of A184 to E of Ω-like loop alone can introduce the cephalosporinase activity in E. coli PBP5 supporting the phenomenon of a single amino acid polymorphism.

Involvement of AmpG in mediating a dynamic relationship between serine beta-lactamase induction and biofilm-forming ability of Escherichia coli.

AmpG permease is implicated both in beta-lactamase induction and peptidoglycan recycling in enterobacterial isolates. Here, physiological studies using molecular genetics show that deletion of AmpG permease dramatically increases beta-lactam susceptibility even in the presence of AmpC, TEM-1 and OXA beta-lactamases. Also, there is an appreciable decrease in the biofilm-forming ability of strains lacking this protein. Expression of this permease in excess probably compromises the integrity of the bacterial cells, leading to cell lysis. Based on these results, we propose that AmpG permease may be used as a potential antibiotic target and its suppression could efficiently inhibit both beta-lactamase induction and biofilm formation.

Glutamate residues at positions 162 and 164 influence the beta-lactamase activity of SHV-14 obtained from Klebsiella pneumoniae.

Extensive production of SHV-14 beta-lactamase makes Klebsiella pneumoniae resistant to beta-lactams. The presence of omega-loop has been reported to influence the beta-lactamase activity, which is also present in SHV-14. Its omega-loop has three glutamates in nearly alternating positions 162, 164 and 167 but their concise role on the behaviour of SHV-14 is unknown. To uncover the influence of each glutamate on SHV-14, we replaced glutamates with alanine and estimated the effect of each mutation by assessing the change in beta-lactam sensitivities in the surrogate Escherichia coli cells and catalytic efficiencies for hydrolysis with the purified proteins. On expression, the clone of wild-type SHV-14 aggravated the resistance of host by 60-500 folds against penicillin and cephalosporin groups of antibiotics. However, the expression of mutated enzymes (especially E164A) substantially reduced the resistance level as compared to the wild type, and the results were in synchrony with the estimated enzymatic efficiencies of wild-type and mutated proteins. Therefore, with further support from the in silico analysis, we hypothesise that mutation at the glutamate residues in the omega-loop of SHV-14 can considerably modulate the beta-lactam sensitivity and hydrolysis, thus revealing the importance of such glutamates as the target for inhibitor design in future.

Use of azidonaphthalimide carboxylic acids as fluorescent templates with a built-in photoreactive group and a flexible linker simplifies protein labeling studies: applications in selective tagging of HCAII and penicillin binding proteins.

This work describes the synthesis of azidonaphthalimide carboxylic acids which act as fluorescent templates with a built-in photoreactive group and a linker thus simplifying the design of protein labeling agents. These were separately connected to selectivity hands like a sulfonamide and ampicillin for successful labeling/detection of HCAII and PBPs, respectively.

A Tyrosine Residue Along with a Glutamic Acid of the Omega-Like Loop Governs the Beta-Lactamase Activity of MSMEG_4455 in Mycobacterium smegmatis.

Mycobacterial beta-lactamases are involved in exerting beta-lactam resistance, though many of these proteins remain uncharacterized. Here, we have characterized MSMEG_4455 of Mycobacterium smegmatis as a beta-lactamase using molecular, biochemical and mutational techniques. To elucidate its nature in vivo and in vitro, and to predict its structure-function relationship in silico analysis is done. The MSMEG_4455 is cloned and expressed ectopically in a beta-lactamase deficient Escherichia coli mutant to establish the in vivo beta-lactamase like nature via minimum inhibitory concentration (MIC) determination. Likewise the in vivo results, purified soluble form of MSMEG_4455 showed beta-lactam hydrolysis pattern similar to group 2a penicillinase. In silico analyses of MSMEG_4455 reveal glutamic acid (E)193 and tyrosine (Y)194 of omega-like loop might have importance in strengthening hydrogen bond network around the active-site, though involvement of tyrosine is rare for beta-lactamase activity. Accordingly, these residues are mutated to alanine (A) and phenylalanine (F), respectively. The mutated proteins have partially lost their ability to exert beta-lactamase activity both in vivo and in vitro. The Y194F mutation had more prominent effect on the enzymatic activity. Therefore, we infer that Y194 is the key for beta-lactamase activity of MSMEG_4455.