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.

Expression of an Extracellular Protein (SMU.63) Is Regulated by SprV in Streptococcus mutans.

In Streptococcus mutans, SprV (SMU.2137) is a pleiotropic regulator that differentially regulates genes related to competence, mutacin production, biofilm formation, and the stress tolerance response, along with some other pathways. In this study, we established a link between SprV and an ∼67-kDa protein in the culture supernatant of strain UA159 that was later confirmed as SMU.63 by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. We discovered that SprV downregulates the transcription and translation of SMU.63. We found that the seven amino acids from the C-terminal region of SprV were also crucial for the expression of SMU.63. Deletion of smu.63 led to increased sucrose-independent biofilm formation and competence. The sprV deletion also increased biofilm formation although this could be partially attributed to the downregulation of smu.63 In an smu.63 sprV double mutant, a synergistic effect was observed in biofilm formation in contrast to effects on competence development. We found that low or excess magnesium ion repressed sprV transcription that, in turn, affected the expression of smu.63 As expected, a magnesium ion-dependent effect of competence and biofilm formation was observed in the UA159 strain. We also replicated the results of SMU.63 expression and competence in S. mutans GS5 that encodes both SprV and SMU.63 homologs and found that the GS5 strain behaves similarly to the UA159 strain, indicating that SprV's effect is strain independent.IMPORTANCE We previously identified a pleiotropic regulator, SprV, in Streptococcus mutans This regulator appears to be highly conserved among streptococci. Here, we showed that SprV regulates the expression of a secreted protein encoded by SMU.63 in S. mutans SMU.63 has been known to impact biofilm formation and genetic competence, two important characteristics that help in colonization of the organism. SMU.63 is also unique since it is known to form amyloid fiber. We found that SprV regulates the expression of SMU.63 at both the transcriptional and translational levels. We also found that the expression of SprV is regulated by magnesium ion concentration. Interestingly, both low and high magnesium ion concentrations affected biofilm formation and genetic competence. Since SMU.63 is also highly conserved among streptococci, we hypothesized that SprV will have a similar effect on its expression.

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.

Postoperative Tinnitus after Vestibular Schwannoma Surgery: A Neglected Entity.

BACKGROUND: This prospective study analyzes the factors responsible for pre and postoperative persistent tinnitus following vestibular schwannoma (VS) surgery and discusses the possible etiopathogenetic mechanisms. MATERIALS AND METHODS: Sixty-seven consecutive patients with unilateral VS operated via the retrosigmoid-suboccipital approach were included in the study. The Cochlear nerve, often unidentifiable from the tumor capsule, was resected during the surgery. Tinnitus Handicap Inventory (THI) score assessed the severity of pre and postoperative tinnitus. RESULT: Twenty-eight (41%) patients had preoperative tinnitus. Out of those 28 patients, 24(85%) had significantly improvement in postoperative THI score. In 15 of the 24 patients, tinnitus subsided completely. In 3 of the 28 (10%) patients, THI scores were unaltered, and in 1 of the 28 (3.5%) patients, THI scores worsened. In 39 (58.2%) patients without preoperative tinnitus, 4 (10%) developed a new-onset postoperative tinnitus. Patients with severe sensory neural hearing loss (SNHL) had significantly higher incidence of postoperative persistent tinnitus (PPT) (P = 0.00) compared to those with mild-to-moderate SNHL. Patients with profound SNHL, however, had a much lower incidence of PPT (P = 0.007; odds ratio = 0. 0.077; 95% CI: 0.009-0.637). Large (P = 0.07) and giant schwannomas (P = 0.03) VS had an increased risk of PPT. Patients with PPT further analyzed with brain stem auditory evoked response (BAER) showed normal contralateral waveform. CONCLUSION: Assessment of tinnitus is mandatory during the management of VS as there are high chances (nearly 46%) of PPT. Preoperative tinnitus, linked to the degree of SNHL (higher incidence in severe SNHL compared to mild-to-moderate/profound SNHL), is dependent on an intact cochlear nerve functioning. However, PPT is dependent on other mechanisms (brain stem/ipsilateral cochlear nuclei compression, and cortical reorganization) as it persists despite cochlear nerve resection.

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.

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.

Factors Leading to Oro-Facial Herpetic Eruptions in Patients Undergoing Surgery for Vestibular Schwannoma.

OBJECTIVE: To study factors influencing oro-facial herpetic eruptions (HEs) in patients undergoing retromastoid suboccipital craniectomy for vestibular schwannomas (VS). METHODS: A retrospective analysis of the prospectively collected database (from July 2014 to December 2015). A total of 87 patients underwent retromastoid suboccipital craniectomy for VS at our center. For the purpose of analysis the patient subset was divided into 2 groups, HE and non-HE. Pearson χ2 test or Fisher exact test were used to identify the factors. RESULTS: The overall incidence of postoperative HE was less than 1% (0.89%, 26 patients of 2916 cases); whereas after VS surgery, it was 20.69% (18 of 87). Demographic profiles of patients in the 2 groups were comparable. Average tumor size (with HE 3.19 ± 2 × 0.67 cm, non-HE 3.38 ± 2 × 1.07 cm), consistency, and laterality also were comparable between the 2 groups. Factors favoring development of postoperative HEs were large size (12 vs. 22, P = 0.013) and preoperative trigeminal nerve (CN V) involvement (9 of 18, 50%, P = 0.046). All patients developed HE in maxillary division of trigeminal nerve (V2), whereas involvement of ophthalmic (V1) and mandibular (V3) divisions were involved less commonly in combination with V2 (V2, 72.2%; V2 + V3, 22.2%; V1 + V2 + V3, 5.6%). The majority of the patients (55.56%) developed HE on postoperative day 3 and none beyond postoperative day 5. All patients responded to empirical oral acyclovir. CONCLUSIONS: The study highlights the relatively high incidence and factors associated with this rare but benign complication.

A putative low-molecular-mass penicillin-binding protein (PBP) of Mycobacterium smegmatis exhibits prominent physiological characteristics of DD-carboxypeptidase and beta-lactamase.

DD-carboxypeptidases (DD-CPases) are low-molecular-mass (LMM) penicillin-binding proteins (PBPs) that are mainly involved in peptidoglycan remodelling, but little is known about the dd-CPases of mycobacteria. In this study, a putative DD-CPase of Mycobacterium smegmatis, MSMEG_2433 is characterized. The gene for the membrane-bound form of MSMEG_2433 was cloned and expressed in Escherichia coli in its active form, as revealed by its ability to bind to the Bocillin-FL (fluorescent penicillin). Interestingly, in vivo expression of MSMEG_2433 could restore the cell shape oddities of the septuple PBP mutant of E. coli, which was a prominent physiological characteristic of DD-CPases. Moreover, expression of MSMEG_2433 in trans elevated beta-lactam resistance in PBP deletion mutants (ΔdacAdacC) of E. coli, strengthening its physiology as a dd-CPase. To confirm the biochemical reason behind such physiological behaviours, a soluble form of MSMEG_2433 (sMSMEG_2433) was created, expressed and purified. In agreement with the observed physiological phenomena, sMSMEG_2433 exhibited DD-CPase activity against artificial and peptidoglycan-mimetic DD-CPase substrates. To our surprise, enzymic analyses of MSMEG_2433 revealed efficient deacylation for beta-lactam substrates at physiological pH, which is a unique characteristic of beta-lactamases. In addition to the MSMEG_2433 active site that favours dd-CPase activity, in silico analyses also predicted the presence of an omega-loop-like region in MSMEG_2433, which is an important determinant of its beta-lactamase activity. Based on the in vitro, in vivo and in silico studies, we conclude that MSMEG_2433 is a dual enzyme, possessing both DD-CPase and beta-lactamase activities.

Transcriptional regulation of Mycobacterium tuberculosis hupB gene expression.

The influence of iron levels on the transcription of the hupB gene in Mycobacterium tuberculosis is the focus of this study. Studies in our laboratory showed HupB to be co-expressed with the two siderophores in low-iron organisms. Mycobactin biosynthesis is repressed by the IdeR-Fe(2+) complex that binds the IdeR box in the mbtB promoter. Recently, we demonstrated the positive regulatory effect of HupB on mycobactin biosynthesis by demonstrating its binding to a 10 bp HupB box in the mbtB promoter. Earlier, we observed that HupB, expressed maximally in low-iron media (0.02 µg Fe ml(-1); 0.36 µM Fe) was still detectable at 8 µg Fe ml(-1) (144 µM Fe) when the siderophores were absent and complete repression was seen only at 12 µg Fe ml(-1) (216 µM Fe). In this study, we observed elevated levels of hupB transcripts in iron-limited organisms. IdeR, and not FurA, functioned as the iron regulator, by binding to two IdeR boxes in the hupB promoter. Interestingly, the 10 bp HupB box, first reported in the mbtB promoter, was identified in the hupB promoter. Using DNA footprinting and electrophoretic mobility shift assays, we demonstrated the functionality of the HupB box and the two IdeR boxes. The high hupB transcript levels expressed by the organism and the in vitro protein-DNA interaction studies led us to hypothesize the sequence of events occurring in response to changes in the intracellular iron concentration, emphasizing the roles played by IdeR and HupB in iron homeostasis.

Iron-regulated protein HupB of Mycobacterium tuberculosis positively regulates siderophore biosynthesis and is essential for growth in macrophages.

Mycobacterium tuberculosis expresses the 28-kDa protein HupB (Rv2986c) and the Fe(3+)-specific high-affinity siderophores mycobactin and carboxymycobactin upon iron limitation. The objective of this study was to understand the functional role of HupB in iron acquisition. A hupB mutant strain of M. tuberculosis, subjected to growth in low-iron medium (0.02 µg Fe ml(-1)), showed a marked reduction of both siderophores with low transcript levels of the mbt genes encoding the MB biosynthetic machinery. Complementation of the mutant strain with hupB restored siderophore production to levels comparable to that of the wild type. We demonstrated the binding of HupB to the mbtB promoter by both electrophoretic mobility shift assays and DNA footprinting. The latter revealed the HupB binding site to be a 10-bp AT-rich region. While negative regulation of the mbt machinery by IdeR is known, this is the first report of positive regulation of the mbt operon by HupB. Interestingly, the mutant strain failed to survive inside macrophages, suggesting that HupB plays an important role in vivo.

Nonscalpel vasectomy as family planning method: a battle yet to be conquered.

Though nonscalpel vasectomy (NSV) technique was introduced in India in 1992 to increase male participation in family planning, it has failed to get adequate momentum and to achieve its goal. We conducted a cross-sectional questionnaire-based survey to get insight into apathy of men towards NSV. The study included 428 respondents. Most of the respondents (97.4%) were aware of NSV as a method for permanent male sterilization. The majority of them (97.2%) knew that NSV is done without any charge and cash incentive is given to the NSV client after the procedure. Though 68.0% respondents agreed that permanent sterilization is a possible option for them, only 34.1% respondents were willing to adopt NSV as a method of family planning. Fear of surgical procedure (40.7%), permanent nature of procedure (22.2%), and religious belief (19.0%) were the common reasons for unwillingness to adopt NSV. We conclude that there is a need to design and develop need-based information, education and communication (IEC) strategy to bridge the existing information gap among the eligible couples regarding NSV to improve its adoption. Involvement of community leaders and satisfied clients and utilization of television and radio would enhance the effectiveness of such interventions.

Serum iron profile and ELISA-based detection of antibodies against the iron-regulated protein HupB of Mycobacterium tuberculosis in TB patients and household contacts in Hyderabad (Andhra Pradesh), India.

BACKGROUND: HupB is a 28 kDa cell-wall-associated protein co-expressed with the siderophores mycobactin and carboxymycobactin in iron-limited Mycobacterium tuberculosis. HupB is expressed in vivo and anti-HupB antibodies are present in the serum of TB patients. METHODS: The aims of this study were to evaluate the serodiagnostic potential of HupB and to correlate levels of anti-HupB antibodies with the serum iron status in TB patients, household contacts and normal healthy controls. RESULTS: TB patients from Hyderabad (India) showed high levels of anti-HupB antibodies compared with household contacts and normal healthy controls. Interestingly, the levels were maximal in extrapulmonary TB patients, with a two-fold higher titre than pulmonary TB patients. Serum iron levels, total iron-binding capacity (TIBC) and percent saturation of serum transferrin were low in subjects with active TB, whilst serum ferritin was notably high in pulmonary TB patients compared with normal controls. CONCLUSIONS: There is a strong negative correlation between serum iron levels and TIBC with the titre of anti-HupB antibodies in subjects with active TB. This study reflects the usefulness of screening for anti-HupB antibodies for diagnosis of pulmonary and extrapulmonary TB in this endemic region.

Simultaneous non-traumatic perforation of the right hepatic duct and gallbladder: an atypical occurrence.

Simultaneous non-traumatic perforation of the extrahepatic bile duct and the gallbladder is an uncommon occurrence that has been infrequently reported. We describe a patient with a spontaneous perforation of both the extrahepatic bile duct and the gallbladder. A contrast-enhanced computed tomography (CECT) scan of the abdomen and endoscopic retrograde cholangiopancreatography (ERCP) demonstrated a perforation of the gallbladder and a free leak from the right hepatic duct, respectively. Endoscopic biliary drainage following a sphincterotomy and biliary stent placement led to a dramatic improvement in the patient's general condition. He was subsequently scheduled to undergo an elective cholecystectomy. Repeat ERCP performed at 4 weeks after the initial stenting showed a normal cholangiogram and a distally migrated stent, which was there after removed. However, early stent removal led to re-perforation of hepatic duct and gallbladder. A repeat endoscopic biliary drainage did not help, and the patient developed biliary peritonitis. Surgical exploration revealed a perforation at the fundus of the gallbladder, 400 ml of biliopurulent collection and a frozen Calot's triangle. A subtotal cholecystectomy, gall stone removal, and a thorough peritoneal lavage were undertaken. The patient improved postoperatively. The second biliary stent was removed after 4 months. This case report highlights the role of endoscopic biliary drainage in the management of an extrahepatic bile duct perforation and warns against the early removal of a biliary stent.